Esterified vasoactive lipids for increasing perfusion pressure of the caruncular arterial bed in mammals

ABSTRACT

Esterified alkanediols which increase perfusion pressure of the caruncular arterial bed in mammals.

GRANT REFERENCE CLAUSE

Work for this invention was funded in part by a grant from United StatesDepartment of Agriculture, Agricultural Research Grant #8901729. TheGovernment may have certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to methods of inducing physiologicaleffects in animals, in particular, a method for decreasing perfusion ofblood through the caruncular arterial bed in mammals.

BACKGROUND OF THE INVENTION

Pinus ponderosa, or western yellow pine (Pinaceae) is abundant inwestern and midwestern states and in western Canada. Needles from P.ponderosa cause cattle to abort if they are consumed during lategestation (McDonald, Veterinary Endocrinology and Reproduction 1969;Stevenson et al., Pine Needle (Pinus ponderosa)-Induced Abortion inRange Cattle, Cornell Vet., pp. 519-524, 1972). P. ponderosa ingestioninduces premature parturition in cattle by causing prolongedvasoconstriction (i.e. increased vascular tone) of the carunculararterial bed partially through increases in potential sensitive calciumchannel (PSC) activity (Christenson et al., Effects of Pine Needles(Pinus ponderosa) by Late-Pregnant Beef Cows on Potential Sensitive Ca²⁺Channel Activity of Caruncular Arteries, J. Reprod. Fertil. pp. 301-306,1993) resulting in a decrease in uterine blood flow.

In pregnancy, blood flow to the gravid bovine uterus increases ≈40 foldfrom conception to term (Ferrell and Ford, Blood Flow, Steroid Secretionand Nutrient Uptake of the Gravid Bovine Uterus, J. Anim. Sci., 50, p.1113-1121, 1980). After day 200, 80-85% of the uterine arterial bloodflows through the caruncular arterial bed (Macowski et al., Distributionof Uterine Blood Flow in the Pregnant Sheep, Am. J. Obstet. Gynecol, 101pp. 409-412, 1968) as a consequence of marked decreases in carunculararterial tone (Ford, Control of Blood Flow to the Gravid Uterus ofDomestic Livestock Species, J. Anim. Sci., pp. 32-43, 1994). Earlycalving following pine needle consumption is accompanied by a profoundconstriction of caruncular arteries and ischemic necrosis at theplacental attachment site (Stuart, et al., Pine Needle abortion incattle: Pathological observations, Cornell Vet. 79, pp. 61-69, 1989). Itis believed that components in pine needles increase the tone of thecaruncular artery, resulting in a reduction of blood flow to thefetal-maternal interface.

Decreases in uterine arterial blood flow appear to result fromactivation of α2-adrenergic receptors on the vascular smooth musclemembrane of the artery (Ford et al., Effects of Ponderosa pine needleingestion on uterine vascular function in late-gestation beef cows, J.Anim. Sci., 70, pp. 1609-1614, 1992). Specifically, α2 activationfacilitates extracellular uptake of calcium via PSC resulting indecreased vessel diameter (i.e. increased vessel tone) and uterine bloodflow.

Pinus ponderosa is the only known species of Pinus to cause abortion incattle in the United States and Canada (Pammel, Manual of PoisonousPlants, The Torch Press p. 330, 1911; James et al., Pine Needle Abortionin Cattle: A Review and Report of Recent Research, Cornell Vet., pp.39-52, 1989; Allison and Kitts, Further studies on the anti-estrogenicactivity of yellow pine needles, J. Anim. Sci., pp. 1155-1159, 1964).This results in large economic losses each year to the beef industry inthe western United States (Lacey et al., Ponderosa Pine: Economic ImpactIn: The ecology and economic impact of poisonous plants on livestockproduction, pp. 95-106, 1988).

Both green and dry needles appear to cause abortion (Jensen et al.,Evaluation of Histopathologic and Physiologic Changes in Cows HavingPremature Births After Consuming Ponderosa Pine Needles, Am. J. Vet.Res. pp. 285-289, 1989), and bark and branch tips appear to containabortifacient principles (Panter et al., Premature Bovine ParturitionInduced by Ponderosa Pine: Effects of Pine Needles, Bark, and BranchTips, Cornell Vet., pp. 329-338, 1990). The nature of the abortifacientprinciples of ponderosa pine needles has been sought for nearly fortyyears. Many test animals or organ assays have been used to suggest anumber of substances ranging from luteolytic agents like theprostaglandins to mycotoxins or the presence of infectiousmicroorganisms (James et al., Pine Needle Abortion in Cattle: A Reviewand Report of Recent Research, Cornell Vet. 79, pp. 39-52 1989).

James and coworkers recently reported that pine needles extracted withselected solvents lost their ability to induce parturition when fed topregnant cows (James et al., Effects of Feeding Ponderosa Pine NeedleExtracts and Their Residues to Pregnant Cattle, Cornell Vet. pp. 33-39,1994). The diterpene known as isocupressic acid was previously isolatedfrom P. ponderosa (Zinkel and Magee, Resin Acids of Pinus ponderosaNeedles, Phytochemistry, pp. 845-848, 1991), and diterpene resins havebeen implicated in embryotoxic effects in mice (Kubic and Jackson,Embryo Resorption in Mice Induced by Diterpine Resin Acids of Pinusponderosa Needles, Cornell Vet. pp. 34-42, 1981).

James et al., found that an 80% pure sample of isocupressic acidisolated from P. ponderosa needles and bark induced early parturition inpregnant cattle (Gardner et al., Ponderosa Pine Needle-Induced Abortionin Beef Cattle: Identification of Isocupressic Acid as the PrincipleActive Compound. J. Agric. Food Chem. pp. 756-761, 1994) thusidentifying one abortifacient principle in ponderosa pine needles. Todate, isocupressic acid is the only compound that has been isolated andsuccessfully shown to cause abortion in pregnant beef cattle.

Numerous classes of natural products have been isolated from P.ponderosa including volatile monoterpenes, sesquiterpenes, diterpenes,and wax alcohols and acids from chitin and suberin. It has now beendiscovered that the lipid diester component from P. ponderosa increasescaruncular arterial tone partially through increasing PSC activity.These lipids may play a role in causing early parturition in pregnantbeef cattle and may potentially be used to cause parturition in othermammals as well.

Accordingly, it is a primary objective of the present invention toprovide a process for preparing novel vasoactive lipid substances whichinduce parturition in mammals.

Another primary objective of the present invention is to prepare a rangeof esterified fatty acids which can be investigated as drugs for theinducement of parturition.

A still further objective of the present invention is to provide a widerange of esterified fatty acids which can be systematically used andtested to determine structure-activity relationships for increasingcaruncular arterial tone partially through increase in PSC activity.

SUMMARY OF THE INVENTION

Vasoactive lipid substances are provided which are isolated from P.ponderosa pine needles. The compounds cause an increase in carunculararterial tone partially through increase in PSC activity, leading tocompounds which may be used to induce parturition in mammals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the low resolution FAB-MS (Matrix 3NBA-Li) of P. ponderosafraction F7(1-10) indicating mixtures of similar compounds.

FIG. 2 shows the 1H-NMR (CDCl₃) of fraction A-1 at 360 MHz.

FIG. 3 shows the 13C-NMR (CDCl₃) of fraction A1 at 90.5 MHz.

FIG. 4 shows the FAB MS spectrum (3NBA) of fraction A1.

FIG. 5 shows the metastable ion spectra of fraction A1 peak m/z596.

FIG. 6 shows the metastable ion spectra of fraction A1 peak m/z624.

FIG. 7 shows the GC total ion current chromatograMS of the neutralfraction obtained by saponification of fraction A1 and derivatizationwith BSTFA and CH₃ OH/BF₃.

FIG. 8 shows the GC total ion current chromatograMS of the acidicfraction obtained by saponification of fraction A1 and derivatizationwith BSTFA and CH₃ OH/BF₃.

FIG. 9 shows the 1H-NMR (CDCl₃) spectra of fraction A3 at 360 MHz.

FIG. 10 shows the 13C-NMR spectra of fraction A3 at 90.5 MHz.

FIG. 11 shows the FAB MS spectrum (3NBA) of fraction A3.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The focus of this application is on the isolation and uses of novelvasoactive esterified fatty acids which are selectively active at theuterine vasculature. Preferred compounds include esters of lauric,myristic and/or palmitic acids. The most preferred compounds are theaforementioned fatty acids esterified with alkanediols. The very mostpreferred compounds are esterified with 1,14-tetradecanedioyl and1,16-hexadecanedioyl. These very most preferred compounds include:

1) 1,14-tetradecanedioyl-dilaurate;

2) 1,14-tetradecanedioyl-1-laurate-14-myristate;

3) 1,14 tetradecanedioyl-dimyristate

4) 1,16-hexadecanedioyl-laurate-myristate;

5) 1,14-tetradecanedioyl-laurate-palmitate;

Other suitable compounds include esters comprised of two fatty acidsesterified together with one alkanediol and one ω-hydroxy fatty acid.These compounds have the following structures: ##STR1##

As previously stated, the parturition-inducing properties of P.ponderosa needles in cattle have been known for many years, but thecomponent or components in the needles responsible for this effect werenot. Isocupressic acid has been isolated and identified as anabortifacient component of P. ponderosa, but so far has been the onlycompound producing this effect which has thusfar been isolated.

Applicants have now successfully isolated a novel class of vasoactivecompounds from P. ponderosa which have never been previously identifiedor isolated. Theoretically, these compounds would have their greatesteffect on the uterine vasculature. During pregnancy, the carunculararterial bed is preferentially dilated so that nutrients from the mothercan more readily reach the fetus. Physiologically, the potentialsensitive calcium channels of the caruncular arterial bed are closed toprevent the influx of calcium which would cause the blood vessels toconstrict. Since blood vessels in the body normally have a high degreeof calcium channel activity, a compound which would increase the influxof calcium would logically have its most dramatic effect on vasculaturewhich is not normally experiencing this severely reduced calcium channelactivity due to the state of pregnancy.

Preliminary observations conducted by Applicants have confirmed thistheory. Based on tests conducted on cattle which have ingested ponderosapine needles, the vasoactive compounds present in the needles appear tobe selectively active for uterine vasculature. After eating the pineneedles, the cattle showed no signs of increased systemic blood pressureor increases in heart rate, but exhibited marked and progressivedecreases in uterine blood flow (Christenson et al., Effects ofIngestion of Ponderosa Pine Needles by Late-Pregnant Cows on UterineBlood Flow and Steroid Secretion, J. Anim. Sci., 70, pp. 531-537, 1992).

Such compounds offer many potential therapeutic uses in mammals.Ponderosa pine needles contain a variety of different mixed chemicalcomponents, some of which induce parturition, but many others which donot and as well may have the potential to produce unwanted side effectsif ingested. Applicants have now identified the chemical structure andcomposition of several compounds present in the pine needles, which whenisolated, purified, and/or synthesized, and fed to mammals producebeneficent and therapeutic results. Further, the possible adverseeffects of the unknown mixed components of pine needles can be avoided.

In addition, due to the unique selectivity of these new compounds foruterine vasculature only, unwanted systemic vascular effects which oftenoccur with vasoactive drugs can be avoided, such as increases in bloodpressure and heart rate.

Potential uses of these new vasoactive compounds include, but are in noway limited to, treatment or prevention of postpartum hemorrhage,induction of labor, and inducement of parturition. Based on the testsconducted, a potential dosing plan for the inducement of in vivoparturition in mammals would be in the broad range of 1/2 to 1milogram/kilogram of body weight/day, orally, for 8-10 days. Thepreferred dose would be 1 milogram/kilogram of body weight/day, orally,for 10 days.

The isolation of the lipid diesters was achieved by the general schemeshown below. Steam distillation, various Soxhlet extraction and acid andbase partitionings of pine needle fractions were all examined during theprocess of developing the most useful protocol for isolating fractionscontaining vasoactive principles.

EXAMPLES Chemicals

Lauric acid, myristic acid, palmitic acid, 10-hydroxydecanoic acid,methyl laurate, methyl myristate, methyl palmitate, dodecan-1-ol,1,12-dodecanediol and 1,14-tetradecanediol were purchased from AldrichCo., Milwaukee, Wis. The purities of each of these compounds and theiridentities were confirmed by chromatography (TLC, GC) and massspectrometry before use.

Plant Material

Pinus ponderosa needles were collected in Custer County, Mont. in theWinter of 1989. A voucher specimen is deposited in the herbarium of thebotany department, Montana State University, Bozeman, Mont.

General Experimental and Equipment

Melting points were recorded on Thomas Hoover Unimelt capillary meltingpoint apparatus and are uncorrected. Infrared (IR) spectra were obtainedusing a Nicolet 205 FT-IR spectrometer connected with a Hewlett-PackardColorPro plotter.

Chemical ionization mass spectral (CIMS) analyses were obtained using aNermag R 1010c instrument. Fast atom bombardment (FAB) experiments wereperformed on a ZAB-HF reversed geometry (BE configuration, where B is amagnetic sector and E is an electrostatic analyzer) mass spectrometer(MS)(VG Analytic, Inc.). It is equipped with an Ion Tech saddle-fieldFAB gun and commercial FAB ion source. Samples were bombarded with 8 keVXe atoMS at an atom gun current of 1.5 mA. 3-Nitrobenzyl alcohol(Aldrich) and magic bullet 5:1 dithiothreitol/dithioerythreitol (Sigma)were the FAB matrices used. Samples were dissolved in methylene chlorideand then 1 μL was added to the matrix on the FAB probe tip (Adams,Analytical Applications and Fundamental Studies, Mass Spectrom. Rev.,pp. 141-186 1990; Jensen and Gross, 1987). Tandem mass spectrometryexperiments (MS-MS) were also performed on the ZAB-HF MS. The techniqueof mass-analyzed ion kinetic energy spectrometry (MIKES) was used todetect the unimolecular ion decompositions in the region between B andE. A particular precursor ion was selected by the magnet and, by ascanning E, product ions that were formed by unimolecular iondecompositions in this region can be observed. The MS-MS spectra are theresult of averaging eight to ten scans using VG software.

Nuclear magnetic resonance (NMR) spectra were obtained on Bruker NM-360MHz and Varian NMR-500 MHz high field spectrometers equipped with an IBMAspect-2000 processor and with a software VNMR version 4.1b,respectively. ¹ H-(360.134 and 499.843 MHz) and ¹³ C-NMR (90.15 and125.697 MHz) spectra were recorded using tetramethylsilane (δ=0) orsolvent peaks as internal standards.

Chromatography

Thin layer chromatography (TLC) was performed on 0.25 mm layers ofsilica gel GF254 (Merck) prepared on 5×20 cm or 20×20 cm glass plateswith a Quikfit Industries spreader (London, UK). Plates were air driedand activated at 120° for a 1 hour prior to use. Plates were developedin a solvent mixture of CH₂ C₁₂ /C₆ H₁₂ /CH₃ CN (20:5:0.1, v/v/v), anddeveloped chromatograms were visualized by spraying with a solution ofH₂ SO₄ :EtOH (1:5 v/v) before warming with a heat gun to develop blackspots. Flash column chromatography (FCC) was performed using JTBakerglassware with 40 mm silica gel (Baker) as the stationary absorbentphase. Solvent compositions similar to those described for TLC were usedin the elution of samples from flash columns.

Pine Needle Fractionation

P. ponderosa pine needles were milled using a Fitzpatrick Model D.Hammer Mill (The Fitzpatrick Co., Elmhurst, Ill.) at medium speed withknives forward and fitted with a #8 mesh stainless steel screen.

For steam distillation, a total of 500 g of milled pine needles weresuspended in 3L dist. H₂ O in a 5 L round bottom flask and refluxed togive 500 mL of distillate. Volatile oils were salted out with NaCl, andextracted with CH₂ C₁₂ (3×250 ml). The organic extract was dried overanhydrous Na₂ SO₄ and concentrated under vacuum to give 700 mg ofvolatile oil. The aqueous phase remaining after distillation wasfiltered, acidified to pH 2 with 6N HCl and extracted with CH₂ C₁₂(3×500 ml). The organic extract was dried over anhydrous Na₂ SO₄ andconcentrated under reduced pressure to obtain 3.5 g of powder. Theacidified aqueous mixture was further extracted with EtOAc/n-BuOH (9:1,v/v)(3×300 ml) to give after drying and concentrating, 3.5 g. Finally,the exhausted pine needles were macerated in 95% EtOH (1.5 L) for threedays. The ethanolic suspension was filtered, the marc was discarded andthe EtOH extract was evaporated under vacuum to five 38 g. Thesefractions were all insoluble in water and soluble in DMSO.

For Soxhlet extractions, 500 g of milled pine needles were exhaustivelyextracted in a Soxhlet apparatus with 2.5 L each of diethyl ether (Et₂O), methylene chloride (CH₂ Cl₂) and methanol (CH₃ OH). Extracts werefiltered and concentrated under reduced pressure to give the followingamounts of samples: Et₂ O, 40 g; CH₂ C₁₂, 12 g; and CH₃ OH, 80 g (Scheme1).

Chromatographic Fractionation of CH₂ Cl₂ Extracts

An 8 g sample of the CH₂ Cl₂ extract was loaded onto a SiO₂ flash column(4 cm×60 cm, 200 g, 40 μ mesh) and the column was eluted with 2 L eachof C₆ H₁₂ ; and mixtures of C₆ H₁₂ +CH₂ C₁₂ and CH₃ OH of increasingpolarities while fractions of 200 mL were collected. Similar fractions(TLC) were combined and concentrated. The largest sample (A) of 5.3 gwas obtained by elution with 20% C₆ H₁₂ in CH₂ Cl₂. This sample (2 g)was further fractionated over a second SiO₂ flash column (2.5 cm×67 cm,75 g, 40 μ mesh silica gel) and eluted with petroleum ether/Et₂ O/HCOOH(50:50:0.8, v/v/v), while 10 ml fractions were collected. The fractionswere monitored by TLC using CHCl₃ /HCOOH (49:1, v/v) and Et2O/petroleumether/HCOOH (50:50:0.8, v/v/v). Fractions 1-10 contained 227 mg of anapparently single spot by TLC at R_(f) 0.72 with CHCl₃ /HCOOH (49:1).However, with CH₂ Cl₂ /C₆ H₁₂ /CH₃ CN (20:5:0.1 v/v/v) as the solventsystem, this fraction actually consisted of a separable mixture of threecomponents of R_(f) 0.25, 0.5 and 0.7.

A simpler chromatographic purification was achieved by adsorbing 1 g ofCH₂ Cl₂ extract onto 80 g of silica gel (40 μmesh) held in a 2.5 cm×60cm column and eluting stepwise with 500 ml of hexane, mixtures of CH₂Cl₂ /C₆ H₁₂ /CH₃ CN beginning with (20:20:0.1, v/v/v) to (20:1:0.1,v/v/v), CH₂ Cl₂ /CH₃ CN (20:0.1, v/v) and with 20 and 50% CH₃ OH in CH₂Cl₂. TLC (CH₂ Cl₂ :C₆ H₁₂ :CH₃ CN, 20:5:0.1, v/v/v) analysis showed thefollowing fraction compositions: fractions 27-33 (A-1), R_(f) 0.5, 8 mg;fractions 34-37 (A-2), 6 mg (mixture); fractions 38-49 (A-3), R_(f) 0.3,19 mg; fractions 64-135, (A04), R_(f) 0.2, 42 mg; fractions 140-475(A-5), a mixture of spots at R_(f) 0.5, 0.15, 0.01 with (CH₂ Cl₂ :C₆ H₁₂:CH₃ CN 20:2:0.5, v/v/v), 117 mg; and fractions 500 or higher (A-6), 487mg (mixture). All fractions except (A-6) and late fractions which elutedwith 20 and 50% CH₃ OH in CH₂ Cl₂ appeared as white crystalline solids.

Sample Hydrolysis, Methylation, O-Trimethylsilylation for GC/MS Analysis

For hydrolysis and derivatization, samples of approximately 1 mg weredissolved in 2 mL of 5% methanolic KOH and refluxed for 10 h. Mixtureswere evaporated to dryness under N₂, 2 mL of H₂ O was added to eachresidue, and the resulting aqueous mixtures were each extracted with CH₂Cl₂ (2×2 mL). Typically, this CH₂ Cl₂ extract was dried over anhydrousNa₂ SO₄ and evaporated to give 0.5 mg of neutral substances. Theremaining aqueous phases were acidified with 2N HCl, extracted with CH₂Cl₂ (2×2 mL), dried over anhydrous Na₂ SO₄ and evaporated to typicallygive 0.4 mg of acidic substances. These neutral and acidic substanceswere subjected to derivatization for GC/MS analysis.

a. Methylation: The acidic fraction after saponification was dissolvedin 0.4 mL of HPLC grade CH₃ OH, mixed with 1 mL BF₃ --CH₃ OH andrefluxed for 2-3 hours. The solvent was removed under N2, 1 mL of dis H₂O was added, and the aqueous mixture was extracted with CH₂ Cl₂ (2×1mL). After drying over anhydrous Na₂ SO₄, rotary evaporation gavemethylated products for GC analysis or further derivatization withBSTFA.

b. O-Trimethylsilylation: This was accomplished by adding 35 μL ofbis(trimethylsilyl)triflouroacetamide (BSTFA) to a solution of 0.7 mg ofneutral substances obtained by saponification in 1 mL CH₂ Cl₂, or 20 μLof BSTFA to a solution of 0.4 mg of methylated acidic compounds in 1 mLof CH₂ Cl₂. The reactants were shaken for 20-30 minutes before sampleswere directly subjected to GC/MS analysis without further workup.

c. GC/MS Analysis: Gas chromatography (GC) was routinely performed witha Hewlett Packard 5890A gas chromatograph equipped with a fused silicacapillary (SPB05) column, 30 m×0.32 mm ID, 0.20 μM film thickness,(Supelco Inc., Bellefonte, Pa.), and linked to a Hewlett Packard 3390Aintegrator. Nitrogen was used as carrier and make up gas at flows of 30and 10 ml/min respectively, and eluting compounds were detected by flameionization detection (FID). Column, injector and detector temperatureswere maintained at 180°-210° C. (5° C./min), 220° and 300° C.,respectively. Both column head carrier gas and hydrogen pressures wereheld at 35 psi. We used this method in evaluating the structures ofhydroxy and oxo-fatty acids derived by the hydration and subsequentoxidation of oleic acid (E1-Sharkawy et al., 1992). Low resolution massspectra were obtained either by direct inlet probe sample admission, orby GC using methyl silicon (DB-1) column (50°-250°, 20° /min) in aTrio-1 mass spectrometer linked with a Hewlett-Packard 5890A Gaschromatograph.

Bioassays

Samples of 5-10 mg of extracts or chromatographic fractions weretriturated into 50 mL of bovine plasma in a glass mortar. Samples werediluted with bovine plasma before being used in the placentome bioassay.

Placentomes were collected at a commercial slaughter facility from thegravid uterine horn of late pregnant cows between 240-270 days ofgestation as determined by measurement of fetal crown rump length.(Evans and Jack, Prenatal Development of Domestic and LaboratoryMammals: growth curves, external features and selected references, Anat.Histol. Embryol. pp. 11-45, 1973). At the laboratory, placentomes wereplaced in an open perfusion chamber which was submerged in oxygenatingKreb's-Ringer solution and the caruncular artery was connected topolyethylene tubing, and the artery was gently perfused to remove bloodfrom the vascular tree. The chamber was then sealed and a Harvardvariable-speed peristaltic pump was used to deliver continuouslyoxygenated Kreb's-Ringer solution at 37° C. The extralumenal flow wasmaintained at 10 mL/min, and the instraluminal flow to each artery wasmaintained at 5 mL/min to achieve intraarterial pressures ofapproximately 80 mm Hg as measured by Statham pressure transducers andrecorded on a Hewlett-Packard 7700 chart recorder. A pulse similar tothat seen in the live animal was imposed on the intra-arterial flow withthe use of a physiological perfusion pump (Medical EngineeringConsultants, Los Angeles, Calif.). A 1/2 hr. equilibration period wasallowed before the start of sample perfusion which allowed theplacentome to establish a constant baseline perfusion pressure.Different Ponderosa pine needle extracts or chromatographic fractionsdissolved in plasma were infused into the caruncular artery preparationat a rate of 0.5 mL/min with a Harvard dual syringe constant infusionpump (Conley and Ford, Effect of Prostaglandin F₂ α-Induced Luteolysison In Vivo and In Vitro Progesterone Production by IndividuralPlacentomes of Cows, J. Anim. Sci., pp. 500-507, 1987). In general, twoplacentomes were perfused first for 20 min with vehicle, then sampleswith increasing concentrations of compounds or extracts for 20 min each,and then vehicle for 20 min. Vascular tone was estimated as theperfusion pressure established by the placentome preparation by the endof each 20 min perfusion period. At the end of each 20 min perfusionperiod (vehicle and samples) perfusion pressure was recorded, and adepolarizing bolus does of 0.2M KCI (200 μL) was injected into theintraluminal flow (5 mL/min) and increased perfusion pressure used toestimate the maximal potential sensitive calcium channel activity aspreviously described (Christenson et al., Effects of Pine Needles (Pinusponderosa) by Late-Pregnant Beef Cows on Potential Sensitive Ca²⁺Channel Activity of Caruncular Arteries, J. Reprod. Fertil., pp.301-306, 1993). Each sample's vasoactivity was determined by bycomparing the measurements of vascular tone and potential sensitivecalcium channel activity at the end of its 20 min perfusion to baselinevalues. Baseline values were determined by averaging values at the endof both 20 min vehicle perfusion periods. Each sample was testedsimultaneously in two placentomes on each experimental day and eachsample was evaluated on at least 2 days.

Extraction and Isolation of Vasoactive Lipids

Steam distillation yielded a relatively small fraction (700 mg, 0.14%yield) of volatile oil. Acid and base partitioning of remainingsuspensions of plant material led to complex extracts, none of whichdisplayed biological activity in the placentome assay system.

For Soxhlet extraction, the best system involved sequential contactswith Et₂ O, CH₂ Cl₂ and CH₃ OH (Scheme 1) to obtain relatively largeyields of pine needle extractables. Et₂ O removes relatively nonpolarsubstances in 8% (w/w) yield, CH₂ Cl₂ gave 2.4% yield of a more polarfraction, while CH₃ OH gave 16% yield (w/w) of the most popular mixtureof components. The total weight extracted represents 26.4% of the dryweight of the plant material used. In all preliminary placentome assays,CH₂ Cl₂ extracts and chromatographic subfractions of the CH₂ Cl₂extracts were active in increasing PSC activity and vascular tone of thecaruncular artery.

The P. ponderosa CH₂ Cl₂ extracts were white, semicrystalline mixtures(TLC) of components. Aqueous acid and base partitioning of CH₂ Cl₂solutions of the extracts gave little indication of the possiblepresence of vasoactive acidic or basic components. Several differenttypes of FCC purifications of this white material were attempted beforesuccessful fractionations were achieved. An apparently TLC pure (R_(f)0.7, with CHCl₃ /HCOOH [49:1]) vasoactive substance designated F7(1-10)was obtained upon initial FCC over silica gel. However, with anothersolvent system (CH₂ CL₂ /C₆ Hl₂ /CH₃ CN, 20:5:0.1 v/v/v), this fractionwas clearly a mixture of three spots migrating at R_(f) 0.2, 0.5, and0.7 which correspond to pure isolated spots later designated A-1, A-3and an unknown material. Using the acetonitrile containing solventsystem, fractions A1-A6 were obtained by FCC (Scheme 1) and subjected tobioassay in the placentome system.

Bioassay Results

The results of placentome bioassays on individual fractions and themixture designated F7(1-10) are presented in Table 1. It was useful toexamine the activity of the F7(1-10) mixture because of the possibilitythat mixtures of compounds could act synergistically or counteract eachother in affecting potential sensitive calcium channel activity and tonewith the placentome system. This sample gave significant increases inboth perfusion pressure (tone) and in potential sensitive calciumchannel activity when perfused through the placentome at 10 μg/mL. Whendiluted to 5 μg/ml, this sample exhibited little effect on tone, but theincrease in potential sensitive calcium channel activity remainedremarkably high (233%). At the lowest dose of 2.5 μg/mL, this fractiongave little response to either measures of vasoactivity.

Fraction A-1 appeared to cause dose-related increases in both tone andpotential sensitive calcium channel activities of the vessels. FractionA-3 increased primarily the KCl response, indicating a more specificeffect on potential sensitive calcium channel activity. Neither fractioncaused increases in vascular tone or potential sensitive calcium channelactivity as high as those observed with F7(1-10). The results suggestthat mixtures of compounds affect both the tone (i.e. vessel diameter)and potential sensitive calcium channel activity of the uterinevasculature, and that the effects of these compounds appear to besynergistic in nature. By spectral and chromatographic analysis, thesesamples contained no terpene acids such as isocupressic acid. FractionsA-4 and A-6 showed little activity, and samples A-2 and A-5 were notassayed because they were mixtures. The bioassay active fractions (A-1and A-3) were subjected to spectral and chemical analysis.

                  TABLE 1                                                         ______________________________________                                        Vasoactivity evaluation of pine needle                                        fractions in the placentome bioassay.                                                μg/mL  % Increase in BPP.sup.a                                                                     % Response to                                  Fraction                                                                             Perfused  at end of 20 min.                                                                           KCl above BPP.sup.b                            ______________________________________                                        F.sub.7 (1-10)                                                                       10        417           362                                                   5         28            233                                                   2.5       28            40                                             A1     10        93            150                                                   5         43            50                                                    2.5       20            20                                             A3     10        50            253                                                   5         22            138                                                   2.5       19            35                                             A4     10        23            5                                              A6     10        20            18                                             ______________________________________                                         .sup.a BPP = Baseline perfusion pressure.                                     .sup.b Measure of potential sensitive calcium channel activity                A2 and A5 were not tested because they were mixtures.                    

LRFAB MS Analysis of Fraction F7(1-10)

Because fraction F7(1-10) was a complex mixture of substances, it wasnot subjected to complete spectral analysis. However, the LRFAB massspectrum of this fraction in 3-NBA saturated with LiI is shown inFIG. 1. Prominent in this spectrum are the ions of m/z 573 (m/z 566+Li),m/z 601 (m/z 594+Li), m/z 629 (m/z 622+Li), and m/z 657 (m/z 650+Li).These ions are the primary components found in fraction A-1. Of lowerintensities are ions of m/z 771 (m/z 764+Li), m/z 799 (m/z 792+Li), m/z828 (m/z 821+Li) and m/z 856 (m/z 849+Li). These ions are the primarycomponents found in fraction A-3. Those found in m/z 970, m/z 998, m/z1026, m/z 1054 and m/z 1082 were the primary components in the bioassayinactive fraction A-4. From this spectrum it is apparent that there areat least four groups of similar components contained in F7(1-10). Therelative amounts of these cannot be directly deduced from the relativeintensities of molecular ions in the spectrum, because each group ofcompounds displays molecular ions of different intensities.

Spectral Analyses of Fraction A-1

Fraction A-1 was apparently pure by TLC. Therefore, it was subjected toIR, 1H-and 13C-NMR and mass spectrometric analysis. The IR spectrumshowed neither sharp nor broad bands between 3000-3600 cm-1, indicatingthe absence of free COOH or OH groups. A strong ester absorption bandoccurred at 1735 cm-1. 1H-NMR (FIG. 2) revealed signals typical forfatty acid esters with a 4-proton triplet at 4.05 ppm (--CH₂ OCO--), a4-proton triplet at 2.28 ppm (--CH₂ --COO--), and 8-proton multiplet at1.6 ppm (--CH₂ --CH₂ --COO--; and CH₂ --CH₂ OCO), a large 56-protonsinglet at 1.26 ppm for numerous overlapping --CH2-- functional groupstypical to those found in fatty acids, and a 6-proton triplet at 0.88ppm representing terminal methyl groups. The 13C NMR spectrum (FIG. 3)exhibited signals for ester carbonyl carbons (174.02 ppm), ether carbons(64.38 ppm), and methyl carbons (14.12 ppm) together with numerousmethylene-group carbon signals.

LRFAB MS with Magic Bullet as matrix (FIG. 4), indicated the presence ofthree major molecular ions of m/z 595.5 (M₁ +H)⁺, 623.5 (M₂ +H)+ and651.6 (M₃ +H)⁺. Confirming results were obtained by cationizing withlithium which resulted in (M+Li)⁺ ions of m/z 601.5, and 629.6, and657.5, respectively. These MS results indicated that thechromotographically pure material was actually a mixture of three majorcompounds and additional minor components. Assuming that relativeintensities of the molecular ions reflected the actual concentrations ofthe three components in Fraction A-1, the major compound was that of m/z623 (48%). Ions of m/z 595 and 651 represented 34% and 18% of FractionA-1. The HRFAB analyses for (M₁ +1)⁺ and (M₂ +1)+ ions resulted inmasses of m/z 595.5674 which corresponds to an elemental composition ofC₃₈ H₇₅ O₄ (theoretical 595.5665), and of m/z 623.5926 which correspondsto elemental composition of C₄₀ H₇₉ O₄ (theoretical 623.5977),respectively. The (M+H)⁺ of m/z 651.6 can be regarded as possessing twoadditional methylene groups in its structure for C₄₂ H₈₃ H₄. Additionalions in the MS indicated that each of the maro components fragment andlose units of 182 u (dalton) which result in the formation of ions ofm/z 413, 441 and 469, respectively. Another major fragmentation pathwayproduces low mass ions of m/z 183, 201, 211, and 229. HRFAB experimentshave confirmed that ions of m/z 183 and 211 can be attributed to theacylium ions of lauric and myristic acid respectively. The identity ofions of m/z 201 and 229 was confirmed by HRFAB analyses to be protonatedlauric and myristic acids, respectively. This MS data suggested thepossibility that the structures of compounds contained in A-1 consistedof fatty acid diesters of alkanediols.

Tandem mass spectrometry experiments (MS-MS) were used to furtheranalyze these compounds. MIKES scans were used to observe andcharacterize the unimolecular ion decompositions of the above (M+H)+ions (AdaMS, Charge-remote fragmentations: Analytical applications andfundamental studies, Mass Spectrom Rev., pp. 141-186, 1990; Jensen andGross, 1987). The precursor ion of m/z 595.5 (FIG. 4) decomposed toproduce ions of m/z 578 via loss of H₂ O, m/z 413 via loss of 183 u orC₁₂ H₂₃ O, and m/z 395 via loss of 201 u or loss of H₂ O from the ion ofm/z 413 (C₁₂ H₂₅ O₂). The low mass product ions include m/z 183, 195,and 201. The fragment ion of m/z 195 is thought to arise by loss of twododecanoic (lauric) acid fragments from a tetradecanediol diester.

The parent ion at m/z 623.6 gave m/z 605 by loss of H₂ O, m/z 441 byloss of an m/z 183 or C₁₂ H₂₃ O, m/z 423 by loss of an m/z 201 fragmentor C₁₂ H₂₃ O₂, m/z 413 by loss of m/z 211 or C₁₄ H₂₇ O, 395 by loss ofm/z 229 or C₁₄ H₂₇ O₂, and m/z 195 by loss of both dodecanoic (lauric)and tetradecanoic (myristic) acids from a tetradecanediol diester.

The precursor ion at m/z 651.6 gave a much more complex and noisy MS/MSspectrum indicating that this ion actually consisted of a mixture ofdifferent compounds possessing the same molecular mass. Usefulinformation from this spectrum showed loss of H₂ O to give m/z 635, andsubsequent fragmentations gave rise to numerous other ions as well.Losses of 183 u for CH₃ (CH₂)₁₀ CO from lauric acid gave m/z 469; of 211u for CH₃ (CH₂)₁₂ CO from myristic acid gave m/z 441; and of 239 u forCH₃ (CH₂)₁₄ CO from palmitic acid gave m/z 413. Ions at m/z 396, 422 and450 can be explained by losses of palmitate (CH₃ (CH₂)₁₄ COOH, m/z 256),myristate (CH₃ (CH₂)₁₂ COOH, m/z 229) and laurate (CH₃ (CH₂)₁₀ COOH, m/z200) from the molecular ion. At the lower end of the MS/MS spectrum,fragment ions for protonated palmitic acid (m/z 257), myristic acid (m/z229) and lauric acid (m/z 201) were observed along with theiraccompanying m/z 239, 211 and 183 fragments, respectively. These resultsconfirmed the presence of these three fatty acids as parts of thestructures in the mixture giving rise to m/z 652. Noteworthy in thisspectrum were the presence of two fragment ions at m/z 195 and 223typical for ions arising from 1,14-tetradecanediol and1,16-hexadecanediol, respectively.

Fraction A-1 was subjected to alkaline saponification in order toconfirm the composition of presumed diesters contained in this mixtureof lipids. Neutral and acidic fractions were trimethylsilylated andmethylated and the derivatized components of A-1 were subjected to GC/MScomparisons with standard compounds (Eglington et al., GasChromotographic-Mass spectrometer Studies of Long Chain HydroxyAcids-III, Organic Mass Spectrometry, pp. 593-611, 1968). Table 2 listsGC retention times and key fragments obtained for fatty acid methylesters and trimethylsilylated alkanols and α,ω-alkanediol standards.From the total ion current chromatogram, and fragmentation patterns, themajor neutral components were identified as 1,14-tetradecanediol and1,16-hexadecanediol (FIG. 8). The acidic fraction (FIG. 7) containedmethyl esters of lauric acid, myristic acid and palmitic acid inrelative proportions of 1:4:1, and 1,14-tetradecanediol and1,16-hexadecanediol in relative proportions of 4:6:1. Peaks with R_(T)at 10.8 min are unknown artifacts that appeared in the GC chromatogramsof both unknown and authentic standards derivatized in this work.

The results from MS and from hydrolysis and derivatization confirmedthat the structures of compounds found in fraction A-1 are esters oflauric, myristic and/or palmitic acids esterfied with1,14-tetradecanediol and/or 1,16-hexadecanediol. The MS/MS resultsconfirmed the presence of 1,14-tetradecanediol-dilaurate(2) and1,14-tetradecanediol-1-laurate-14-myristic(3) for molecular ions at m/z595 and 623. The ion at m/z 651 is a mixture of compounds of isomers.Based upon saponification and MS/MS analyses, there are only threepossible combinations of fatty acid esters with alkanediols that matchm/z 651. These are 1,14-tetradecanediol-dimyristate(4),1,16-hexadecanedioyl-laurate-myristic(6) and1,14-tetradecanediol-laurate-palmitate(5).

A separate isolation and analysis of A-1 yielded a mass spectrumslightly different than that originally seen for A-1. The major ions atm/z 595, 623, and 651 were evident albeit in different proportions thanthe previous A-1 fraction. A new ion at m/z 568 consistent for thestructure 1,12-dodecanedioyl-dilaurate (C₃₆ H₇₀ O₄)(1) was observed.MS/MS of this ion gave m/z 550 (C₃₆ H₆₈ O₃) by loss of H₂ O, m/z 385 byloss of m/z 183 or C₁₂ H₂₃ O, m/z 367 by loss of m/z 201 or C₁₂ H₂₃ O₂,and m/z 168 for C₁₂ H₂₄ derived from 1,12-dodecanedioyl-dilaurate whicheliminates the two ester fragments. This result confirmed the presenceof the three compounds identified earlier in fraction A-1, and anadditional derivative dodecanedioyl-dilaurate(1) in a second A-1fraction. The results also underline the complexities in isolatingmixtures of highly similar lipid substances from pine needle extracts.The structures of the compounds identified in the A-1 fraction are:##STR2##

                  TABLE 2                                                         ______________________________________                                        GC-Retention times and key MS fragments of                                    fatty acid methyl esters, trimethylsilylated                                  alkanols, α, ω-alkanediols, and TMS-ω-                      hydroxyhexadecanoic acid methyl ester.                                                    M/Z     GC-Retention                                                                             Key MS Fragments                               COMPOUND    (M+)    time       M/Z                                            ______________________________________                                        (Methyl Esters)                                                               Lauric(C-12)                                                                              214     7.75       183, 74                                        Myristic(C-14)                                                                            242     8.98       211, 74                                        Palmitic(C-16)                                                                            270     10.11      239, 74                                        (TMS Derivatives)                                                             Dodecan-1-ol                                                                              258     8.12       243, 227, 185,                                                                168, 75, 73                                    Tetradecan-1-ol                                                                           286     9.28       271, 255, 213,                                                                196, 75, 73                                    Hexadecan-1-ol                                                                            314     10.35      299, 283, 224                                                                 75, 73                                         Octadecan-1-ol                                                                            342     11.34      327, 311, 75, 73                               1,12-Dodecanediol                                                                         346     10.05      315, 241, 75, 73                               1,14-Tetra- 374     11.14      343, 284, 269,                                 decanediol                     75, 73                                         (TMS, Methyl Ester)                                                           16-Hydroxyhexa-                                                                           358     11.2       343, 284, 269, 75                              decanoic acid                                                                 ______________________________________                                    

Spectral Analysis of Fraction A-3

A-3 was the second chromatographically pure (TLC(CH₂ Cl₂ /C₆ H₁₂ /CH₃ CN(20:5:0.1 v/v/v) fraction isolated from the CH₂ Cl₂ pine needle extract(Scheme 1). This fraction was less active in elevating tone in theplacentome assay (Table 1) but was considerably more active in elevatingthe KCl response. The infrared, 1H- (FIG. 9) and ¹³ C-NMR (FIG. 10)spectra were essentially the same as for A-1. There were no free OH orCOOH groups apparent and a prominent ester absorption band was observed.In the ¹ H-NMR spectrum, differences were found in the relative protonintegrations for signals grouped at 4.05 ppm, 2.28 ppm, 1.60 ppm, 1.26ppm and 0,88 ppm. Interestingly, the triplet signal at 0.88 ppm, whichrepresents end-chain methyl groups, was smaller than it was in the ¹H-NMR spectrum of A-1. The ¹ H-NMR spectrum also indicated thatrelatively simple and terminally oxygenated alkanes and fatty acids werelikely components of fraction A-3. The ¹ H-NMR results would rule outthe presence of 2° alcohols or ether functional groups in the componentsof this fraction. The ¹³ C-NMR spectrum clearly indicated the presenceof ester, numerous methylene, --O--CH₂ --CH₂ -- and terminal methylgroup signals like A-1.

For A-3, LRFAB (Magic Bullet)(FIG. 11) gave ions of m/z 793.7 (M₁ +H)+,821.7 (M₂ +H)+, 849.7(M₃ +H) and 877.8 (M₄ +H)+. Mass spectral resultsindicated that A-3 was a mixture of at least four major compounds eachapparently differing in mass by two CH₂ -- units from one another. HRFABin 3-NBA (Magic Bullet) matrix gave m/z 821.7548 for C₅₂ H₁₀₁ O₆(M+1)+(theoretical 821.7597). This compound differs in mass by C₁₂ H₂₂O₂ verses the major compound in fraction A-1 at m/z 623 for C₄₀ H₇₉ O₄and identified as 1,14-tetradecanedioyl-laurate-myristate. Thedifference in structure can be explained by the presence of an ω-hydroxyfatty acid.

Saponification of A-3 and GC/MS of the resulting O-trimethylsilylatedneutral CH₂ Cl₂ extract indicated that 1,14-tetradecanediol-di-O-TMS wasthe major (90%) neutral component with about 10% of1,16-hexadecanediol-1-O-TMS. These results were identical to thoseobtained with authentic compounds, and seen earlier with fraction A-1(FIG. 7). The acidic extract of A-3 gave total ion chromatogram peaksand fragmentation patterns consistent with the presence of methylmyristate, methyl palmitate, O-trimethylsilyl-ω-OH-hexadecanoic acidmethyl ester, 1,14-tetradecanediol-di-O-TMS, and1,16-hexadecanediol-1-O-TMS (Table 2). The major difference in theacidic fraction of A₃ was the presence of a major peak (@90% ofacids)(T_(R) 11.2 min) matching with the O-TMS derivative of16-hydroxy-hexadecanoic acid methyl ester with ions of m/z 343=(M-15),284=(M-74), 269=(M-15-74), 75, 74 and 73.

MS data and derivatization of saponified samples suggest the presence ofmuch more complex components within the A-3 mixture of compounds. Themajor diol is 1,14-tetradecanediol, and palmitic and myristic acidsappear to be common fatty acid components of compounds in A-3. Thepresence of ω-hydroxy-hexadecanoic acid (16-hydroxypalmitic acid)suggests that the structures of esters found in A-3 are comprised of twofatty acids esterfied together with one alkanediol and one ω-hydroxyfatty acid. We suggest the structures of compounds 7-11, previouslyshown, that fit the MS and chemical derivatization GC/MS data. Thecompound with m/z 821 is represented as structure 10 for C₅₂ H₁₀₁ O₆. Inall but the compound with the lowest mass at m/z 792, ω-hydroxypalmiticacid (16-hydroxyhexadecanoic acid) exists as the hydroxy-fatty acidcomponent of the esters. In order to obtain m/z 792,14-hydroxytetradecanoic acid replaces 16-hydroxyhexadecanoic acid shownin all other structures, even though direct evidence for its presence insaponified samples was not seen. Compounds with m/z 876 can berepresented by more than one isomeric form such as 7 or 8. Attempts toderive confirming information by MS/MS analysis of peaks for fractionA-3 were unsuccessful.

This work describes new structural classes of vasoactive lipidsdominated by the presence of alkanediols esterified with myristic and/orlauric acids. The fact that components of A1 and A3 appear to havedifferential effect on vascular tone and potential sensitive calciumchannel activity suggests that reductions in blood flow in vitro andperhaps in vivo may be due to the presence of structurally similarcompounds with two types of vasoactivity.

As shown above, several of the new compounds isolated by Applicantsdemonstrated ability in increasing the perfusion pressure in placentomebioassays. The components present in the F7(1-10) fraction, whichcontain compounds present in both the A1 and A3 fractions, caused thehighest increase in both vascular tone and potential sensitive calciumchannel activity, suggesting that the A1 and A3 fractions producesynergistic effects when used in combination. The isocupressic acidcomponent previously isolated by James et al. was not present in any ofthese fractions.

Therefore it has been demonstrated that the invention disclosed aboveaccomplishes at least all of its stated objectives.

In the specification there has been set forth examples which are notmeant to limit the invention in any manner but are provided fordemonstrative purposes only.

What is claimed is:
 1. A method of increasing the perfusion pressure ofcaruncular arterial beds in mammals, comprising: administering a small,but perfusion pressure-increasing, amount of a substantially pureesterified fatty acid, wherein the fatty acid is selected from the groupconsisting of lauric, myristic and palmitic acid.
 2. The method of claim1 wherein the fatty acid is esterified with an alkanediol.
 3. The methodof claim 1 wherein the alkanediol is 1,14-tetradecanediol.
 4. The methodof claim 1 wherein the alkanediol is 1,16-hexadecanediol.
 5. The methodof claim 1 wherein the esterified fatty acid is selected from the groupconsisting of:1,14-tetradecanedioyl-dilaurate;1,14-tetradecanedioyl-1-laurate-14-myristate;1,14-tetradecanedioyl-dimyristate;1,16-hexadecanedioyl-laurate-myristate; and1,14-tetradecanedioyl-laurate-palmitate.
 6. The method of claim 1wherein the esterified fatty acid is 1,12-dodecanedioyl-dilaurate. 7.The method of claim 1 wherein the esterified fatty acid is selected fromthe group consisting of: ##STR3##
 8. The method of claim 5 wherein thedose is 1/2 to 1.5 mg/kg of body weight/day, orally for 8-10 days. 9.The method of claim 8 wherein the dose is 1.5 mg/kg of body weight/day,orally, for 10 days.
 10. A method of increasing the perfusion pressureof caruncular arterial beds in mammals, comprising: administering asmall, but perfusion pressure-increasing, amount of a substantially purefatty acid esterified with a compound selected from the group consistingof 1,14-tetradecanedioyl, 1,12-dodecanedioyl, and 1,16-hexadecanedioyl,wherein said fatty acid is selected from the group consisting of lauric,myristic, and palmitic acid.
 11. A method of increasing the perfusionpressure of caruncular arterial beds in mammals,comprising:administering a small, but perfusion pressure-increasing,amount of a substantially pure esterified fatty acid selected from thegroup consisting of: 1.14-tetradecanedioyl-dilaurate;1,14-tetradecanedioyl-1-laurate-14-myristate;1,14-tetradecanedioyl-dimyristate;1,16-hexadecanedioyl-laurate-myristate;1,14-tetradecanedioyl-laurate-palmitate; 1,12-dodecanedioyl-dilaurate;1-(16-laurylatepalmitate)-14-myristate-tetradecanediol;1-(16-laurylatepalmitate)-16-laurate-hexadecanediol;1-(16-laurylatepalmitate)-14-laurate-tetradecanediol;1-(16-laurylatepalmitate)-12-laurate-dodecanediol; and1-(14-laurylatemyristate)-12-laurate-dodecanediol or combinationsthereof.